Receptor cDNA Research Articles

The Cell Surface Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Contributes to Epidermal Growth Factor Receptor-mediated Cell Migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Construction of Response Vectors in Yeast for Environmental Estrogen Bioassay

The environmental estrogen can be measured in modified yeast cells by transcription activation of reporter genes. To establish such yeast estrogen system, the full-length human estrogen receptor (hER) cDNA and estrogen responsive elements (EREs) linked to the upstream of CYC1 promoter were cloned into the yeast expression vector via pPIC9 vector respectively to construct the response vectors named as pER-PIC9 (E-9) and pERE2-CYC-PIC9K-EGFP (EC-9-E), which were transformed into Pichia pastoris at the same time for detecting environmental estrogens based on yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein. In the present study, we developed a rapid yeast estrogen bioassays by creating stably transfected strains with EGFP as a measurable reporter protein.

Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits

P2 receptors are involved in the regulation of ocular physiological processes like intraocular pressure (IOP). In the present study, the involvement of P2Y(2) receptors in the hypertensive effect of nucleotides was investigated by use of antagonists and of a siRNA designed for the P2Y(2) receptor. Agonists of the P2Y(2) receptor a as well as P2 antagonists were applied to eyes of New Zealand rabbits, and the changes in IOP were followed for up to 6 h. Cloning of the P2Y(2) receptor cDNA was done using a combination of degenerate reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). siRNA was synthesized and tested by immunohistochemistry. Single doses of 2-thioUTP, UTP-γ-S and UTP increased IOP. This behaviour was concentration-dependent and partially antagonized by reactive blue 2. Silencing the P2Y(2) receptor was observed in the ciliary body by immunohistochemistry labelling, where a reduction in the immunofluorescence was observed. This reduction in the expression of the P2Y(2) receptor was concomitant with a reduction in IOP, which was measurable 24 h after treatment with the siRNA, maximal after 2 days, followed by a slow increase towards control values for the following 5 days. Application of the P2Y(2) agonists after pretreatment of the animals with this siRNA did not produce any change in IOP. P2Y(2) receptors increase IOP in New Zealand rabbits. The application of a siRNA for this receptor significantly reduced IOP, suggesting that this technology might be used for the treatment of glaucoma.

Functional Characterization of Hypertrehalosemic Hormone Receptor in Relation to Hemolymph Trehalose and to Oxidative Stress in the Cockroach Blattella germanica

Hypertrehalosemic hormone (HTH) is a peptide hormone that belongs to the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family, which exerts pleiotropic actions related to catabolic reaction and stress response. AKH peptides have been demonstrated to participate in stress response including oxidative stress in several insects. In order to study the signaling pathway of HTH involved in anti-oxidative stress, we have characterized a HIH receptor cDNA in Blattella germanica (Blage-HTHR) in structural and in functional terms using RNA interference (RNAi). Blage-HTHR is expressed in various female adult tissues (brain–CC–CA, ventral nerve cord, midgut, fat body, oviduct), but maximal expression is observed in the fat body. RNAi-mediated knockdown of Blage-HTHR expression results in a significantly lower level of hemolymph trehalose, even though HTH is exogenously administered. Paraquat elicits lethal oxidative stress in B. germanica, and co-injection of paraquat and HTH reduces this detrimental effect and extends the median survival time. Interestingly, the “rescue” effect of HTH on mortality caused by paraquat is diminished in specimens with depleted expression of Blage-HTH and Blage-HTHR. Finally, lipid peroxidation in the hemolymph increases 4 h after paraquat treatment, in comparison with control specimens or with HTH-treated specimens. However, lipid peroxidation induced by paraquat was not “rescued” by HTH in Blage-HTH and Blage-HTHR knockdown specimens. Our results demonstrate that HTH acts as a stress hormone mediating anti-oxidative protection in B. germanica, and that its receptor, Blage-HTHR, is essential for this action.

Histamine 3 Receptor Activation Reduces the Expression of Neuronal Angiotensin II Type 1 Receptors in the Heart

In severe myocardial ischemia, histamine 3 (H₃) receptor activation affords cardioprotection by preventing excessive norepinephrine release and arrhythmias; pivotal to this action is the inhibition of neuronal Na⁺/H⁺ exchanger (NHE). Conversely, angiotensin II, formed locally by mast cell-derived renin, stimulates NHE via angiotensin II type 1 (AT₁) receptors, facilitating norepinephrine release and arrhythmias. Thus, ischemic dysfunction may depend on a balance between the NHE-modulating effects of H₃ receptors and AT₁ receptors. The purpose of this investigation was therefore to elucidate the H₃/AT₁ receptor interaction in myocardial ischemia/reperfusion. We found that H₃ receptor blockade with clobenpropit increased norepinephrine overflow and arrhythmias in Langendorff-perfused guinea pig hearts subjected to ischemia/reperfusion. This coincided with increased neuronal AT₁ receptor expression. NHE inhibition with cariporide prevented both increases in norepinephrine release and AT₁ receptor expression. Moreover, norepinephrine release and AT₁ receptor expression were increased by the nitric oxide (NO) synthase inhibitor N(G)-methyl-L-arginine and the protein kinase C activator phorbol myristate acetate. H₃ receptor activation in differentiated sympathetic neuron-like PC12 cells permanently transfected with H₃ receptor cDNA caused a decrease in protein kinase C activity and AT₁ receptor protein abundance. Collectively, our findings suggest that neuronal H₃ receptor activation inhibits NHE by diminishing protein kinase C activity. Reduced NHE activity sequentially causes intracellular acidification, increased NO synthesis, and diminished AT₁ receptor expression. Thus, H₃ receptor-mediated NHE inhibition in ischemia/reperfusion not only opposes the angiotensin II-induced stimulation of NHE in cardiac sympathetic neurons, but also down-regulates AT₁ receptor expression. Cardioprotection ultimately results from the combined attenuation of angiotensin II and norepinephrine effects and alleviation of arrhythmias.

Insect insulin receptors: insights from sequence and caste expression analyses of two cloned hymenopteran insulin receptor cDNAs from the fire ant

The insulin and insulin-like growth factor (IGF) signalling (IIS) pathway in the honey bee (Apis mellifera) is linked to reproductive division of labour and foraging behaviour. Two insulin receptor genes are present in the released genomes of other social hymenopterans. Limited information is available on the IIS pathway role in ants. The predicted insulin receptor sequences from the recently released draft genome of the fire ant Solenopsis invicta (Hymenoptera: Formicidae) are incomplete and biologically significant data are also lacking. To elucidate the role of the IIS pathway in the fire ant, two putative insulin receptors (SiInR-1 and SiInR-2) were cloned; the first InR cDNAs cloned from social insects. Analyses of putative post-translational modification sites in SiInRs revealed the potential for differential regulation. We investigated the transcriptional expression of both receptors at different developmental stages, castes and queen tissues. In last instar larvae and pharate pupae of workers and reproductive, transcriptional abundance of both receptors was negatively correlated with body size and nutritional status. The expression level of both receptors in different queen tissues appears to correlate with requirements for queen reproductive physiology and behaviours. This study contributes new information to the understanding of social insects because in fire ants juvenile hormone acts as a gonadotropin and workers are fully sterile, contrary to honey bees.

Orbital fibrosis in a mouse model of Graves' disease induced by genetic immunization of thyrotropin receptor cDNA

The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered by in vivo skeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. Female BALB/c mice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD.

Novel Neuropeptides Involved in the Control of Puberty and Reproduction in Fish

Introduction: The findings that inactivation of kisspeptin signaling, because of mutations in the kisspeptin receptor, is associated with hypogonadotropic hypogonadism and absent or delayed puberty in man [1] stimulated the many subsequent studies that together have led to a new view of the neural control of GnRH release [2]. With this in mind, the recent observations [3] that mutations of the genes encoding neurokinin B (NKB) (TAC3) and its receptor (TAC3R; NKBR) were also associated with hypogonadotropic hypogonadism, is therefore of considerable interest. In order to study the physiological function(s) and evolutionary conservation of NKB, we cloned tac3 and tac3 receptor cDNAs from several fish species, including zebrafish. Methods: Zebrafish tac3a, tac3b, tac3Ra and tac3Rb were cloned by synteny, according to Biran et al. [4]. A phylogenetic tree of all the currently known vertebrate neurokinin genes was generated with the neighborjoining method. Real-time PCR and receptor transactivation assays were performed as previously described [4]. Results and Discussion: A comparison of the zebrafish protein coding sequences of tac3 cDNAs with those of the human and mouse TAC3 proteins showed identities of 23% and 24%, respectively. The phylogenetic tree showed that the vertebrate neurokinin genes fall into two distinct lineage groups. One lineage includes the mammalian and rodent TAC3, and all the piscine Tac3 that were cloned in the present study. The second lineage includes the mammalian and fish tac1. Nonetheless, high identity was found between different fish species, in the region encoding the NKB; all shared the common C-terminal sequence. Many genes encoding the tachykinins have been found to encode a precursor that produces more than one tachykinin. However, in mammals TAC3 is unusual in that it encodes only a single tachykinin – the NKB. Interestingly, we have found that in zebrafish, exon 3 of both tac3s encodes a well conserved additional tachykinin. Furthermore, in silico analysis revealed that this new tachykinin exists in all other fish species whose genomes are known, and it was termed NKF. NKF also possesses the common C-terminal sequence FVGLM, known as the tachykinin signature. zftac3a-expressing neurons were localized in specific brain nuclei that are known to be implicated in reproduction, whereas zftac3b-expressing neurons were more dispersed throughout the brain. Zebrafish tac3a – but not tac3b – mRNA levels gradually increased during the first few weeks of life, and peaked in fish with ovaries containing mature oocytes or with testes containing mature spermatozoa. Estrogen treatment of mature fish causes increase in tac3a, kiss2 and kiss1rb expression in males, with no significant change in females. Tac3Ra and tac3Rb transduce their activity via both PKC/Ca and PKA/cAMP pathways. Both tac3 receptor types were very sensitive to amidation of their cognate ligands. Conclusion: These results indicate that the NKB/NKBR system may participate in puberty initiation in fish. Moreover, this novel system may be involved, in parallel with the kisspeptin system, in neuroendocrine regulation of GnRH secretion.

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.

Evolution of Three Human GPCRs for Higher Expression and Stability

We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target—the tachykinin receptor NK1, which is hardly expressed in E. coli and cannot be functionally solubilized—receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein.

Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7–3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7–3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7–3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5 h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.

Ghrelin Receptor in Two Species of Anuran Amphibian, Bullfrog (Rana catesbeiana), and Japanese Tree Frog (Hyla japonica)

We have identified cDNA encoding a functional growth hormone secretagogue-receptor 1a (GHS-R1a, ghrelin receptor) in two species of anuran amphibian, the bullfrog (Rana catesbeiana), and the Japanese tree frog (Hyla japonica). Deduced receptor protein for bullfrog and Japanese tree frog (tree frog) was comprised of 374- and 371-amino acids, respectively. The two receptors shared 86% identity, and are grouped to the clade of the tetrapod homologs by phylogenetic analysis. In functional analyses, ghrelin and GHS-R1a agonists increased intracellular Ca2+ concentration in GHS-R1a-transfected-HEK293 cell, but ligand selectivity of ghrelin with Ser3 and Thr3 was not observed between the two receptors. Bullfrog GHS-R1a mRNA was mainly expressed in the brain, stomach, and testis. In the brain, the gene expression was detected in the diencephalon and mesencephalon, but not in the pituitary. Tree frog GHS-R1a mRNA was predominantly expressed in the gastrointestinal tract and ovary, but not detected in the pituitary. In bullfrog stomach but not the brain, GHS-R1a mRNA expression increased after 10 days of fasting. For tree frog, GHS-R1a mRNA expression was increased in the brain, stomach and ventral skin by 10 days of fasting, and in the stomach and ventral skin by a dehydration treatment. Intracerebroventricular injection of ghrelin in dehydrated tree frog did not affect water absorption from the ventral skin. These results suggest that ghrelin is involved in energy homeostasis and possibly in osmoregulation in frogs.

Glutamate-mediated signaling in Schistosoma mansoni: A novel glutamate receptor is expressed in neurons and the female reproductive tract

l-Glutamate is a major neurotransmitter of both vertebrates and invertebrates. Earlier studies have shown that glutamate stimulates neuromuscular activity in the bloodfluke, Schistosoma mansoni, but its mode of action is unknown. Here we describe a novel glutamate receptor in S. mansoni (SmGluR), the first of its kind to be identified in a parasitic flatworm. SmGluR belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to metabotropic glutamate receptors from other species. The full-length receptor cDNA was cloned, stably expressed in HEK-293 cells and shown to be activated by glutamate, whereas aspartate and the glutamate derivative, gamma-aminobutyric acid (GABA) had no significant effect. Among the classical (mammalian) agonists and antagonists tested, only LY341495 was able to interact with the schistosome receptor, suggesting that the pharmacological profile of SmGluR is substantially different from that of receptors in the host. The presence of SmGluR in the parasite was verified by immunoprecipitation and Western blot analyses, using a specific peptide antibody. Confocal immunolocalization studies revealed that SmGluR is strongly expressed in the nervous system of adult worms and larvae. In the adults, the receptor was detected in the longitudinal nerve cords and cerebral commissures, as well as the peripheral nerve fibers and plexuses innervating the acetabulum and the somatic musculature. Outside the nervous system, SmGluR was detected along the length of the female reproductive system, including the oviduct, ootype and the uterus. A comparative expression analysis at the RNA level revealed that SmGluR is expressed at about the same level in cercaria and adult stages, as determined by quantitative reverse-transcription PCR. The results identify SmGluR as an important neuronal receptor and provide the first molecular evidence for a glutamate signaling system in schistosomes.

Elevated GnRH receptor expression plus GnRH agonist treatment inhibits the growth of a subset of papillomavirus 18‐immortalized human prostate cells

Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated. Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis. High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists. Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists.

POMD11 Cannabinoids are neuroprotective in a human cell culture model of Parkinson's disease

BackgroundWe have previously shown a protective effect of Δ9-tetrahydrocannabinol (THC) in Parkinson's disease (PD) cell culture models associated with increased CB1 cannabis receptor cDNA. Here we investigate whether this protective...

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Pathways of angiogenic resistance in glioblastoma: Adaptations in growth factor signaling in response to VEGF and HGF attenuation

Abstract Background: Glioblastoma is the most common and lethal central nervous system malignancy with a median survival often less than 18 months. Despite promising initial responses to anti-angiogenic therapies many patients with this disease develop resistance to treatment via poorly understood mechanisms. Here we demonstrate that glioblastoma cell lines grown under growth factor antagonizing conditions exhibit an altered phenotype with changes in growth factor signaling, gene expression as well as in vivo tumorigenesis. Methods: U87 cells were cultured under constant pressure with bevacizumab (3.0 mg/mL), AMG102 (1.0 µg/mL), or a combination of both drugs long term (seven or more passages) in adherent culture. In vitro expression analysis was carried out by growth factor and receptor tyrosine kinase arrays, cDNA microarray, and ELISA. Intracranial xenograft studies demonstrated resultant differences in growth kinetics and survival. Results: Hepatocyte growth factor (HGF), glial derived neurotrophic factor (GDNF), and platelet-derived growth factor AA (PDGFAA) were elevated at 5.3, 4.5, and 3.9 fold in combinatorially treated cells versus controls, respectively. These changes correlated with relative increases in corresponding receptor mRNA and protein expression. Furthermore, AMG102 and combinatorially pressured cells were highly tumorigenic as compared to control and bevacizumab treated cells in vivo (Median survival 10 ± 1 vs. 32 ± 2 days, respectively). Conclusion: Herein we describe concomitant increases of various oncogenic growth factors which might potentiate tumor survival and therapeutic resistance. These results suggest several potential molecular bypass mechanisms facing proposed angiogenic strategies. Evaluation of resistant phenotypes provides insight on the adaptive complexity of glioblastoma and offers potential for targeting recurrent disease.

Progesterone receptor gene expression in the accessory sex glands of veal calves

This study investigated progesterone receptor (PR) cDNA expression in the testes, prostate and bulbourethral glands of prepubertal calves treated experimentally with high and low doses of 17β-oestradiol and with testosterone....

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2 and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment.